Treatment of epilepsy

ABSTRACT

The present invention discloses isoquinolines and 1H-2-Benzopyranes and their use in the treatment and prevention in epilepsy and other seizures. The present invention further discloses methods to screen isoquinoline- and 1H-2-Benzopyran-like molecules as pharmaceutically active compounds.

FIELD OF THE INVENTION

The present invention provides a pharmaceutical composition for treatingepilepsy.

BACKGROUND OF THE INVENTION

Epilepsy is among the most common severe neurological conditions,affecting more than 70 million people worldwide [Sander (2003) Curr OpinNeurol 16, 165-170; Ngugi et al. (2010) Epilepsia 51, 883-890; Singh andTrevick (2016) Neurol Clin 34, 837-847]. It is characterized by anenduring predisposition of the brain to generate epileptic seizures,with neurobiologic, cognitive, psychological, and social consequences[Fisher et al. (2005) Epilepsia 46, 470-472.]. The treatment of epilepsyconsists mostly of pharmacotherapy with antiseizure drugs (ASDs) tocontrol seizures [Golyala & Kwan (2017) Seizure 44, 147-156.]. Despiteconsiderable efforts, current ASDs fail to control the seizures of 30%of patients due to drug-resistance [Dalic & Cook (2016) NeuropsychiatrDis Treat 12, 2605-2616.]. Uncontrolled epilepsy can result in a poorerquality of life because of physical, psychological, cognitive, social,and occupational problems [Golyala, A., and Kwan, P. (2017) Seizure 44,147-156; Blond et al. (2016) Neurol Clin 34, 395-410, viii]. Moreover,first-line ASDs are associated with important adverse effects that cansignificantly impact daily life and are a main cause of treatmentfailure [Dalic & Cook (2016) Neuropsychiatr Dis Treat 12, 2605-2616;Moshe et al. (2015) Lancet 385, 884-898; Cramer et al (2010) Expert RevNeurother 10, 885-891].

Hence, there is a high need for the development of safer ASDs that aremore effective against drug-resistant seizures.

Zebrafish animal models for screening compounds for anti-epilepticactivity has been described [e.g. MacRae & Peterson (2015) Nat Rev DrugDiscov 14, 721-731; Crawford et al. (2008) Planta Med 74, 624-632]

SUMMARY OF THE INVENTION

The present invention discloses isoquinoline alkaloids which wereisolated from the bioactive marine-derived fungus Aspergillus insuetus.The compounds were investigated for antiseizure activity in the larvalzebrafish PTZ seizure model. TMC-120A and TMC-120B were found to haveantiseizure activity. The antiseizure activity of TMC-120A and TMC-120Btranslated to a mouse model of drug-resistant focal seizures. Followingstructural analogues of TMC-120A and TMC-120B are candidate compoundsfor antiseizure activity in the larval zebrafish PTZ seizure model:TMC-120C, penicisochroman G, ustusorane B,7-methylfuro[3,2,h]isoquinoline-3(2H)-one,7-methylfuro[3,2-h]isoquinoline-3(2H)-one (compound 6),2-(7-methyl-2,3-dihydrofuro[3,2-h]isoquinoline-2-yl)-propan-2-ol(compound 7).

Based on the prominent antiseizure activity in zebrafish, the presentinvention relates to TMC-120A, TMC-120B, TMC-120C, penicisochroman G,ustusorane B, 7-methylfuro[3,2,h]isoquinoline-3(2H)-one, compound 6, andcompound 7 as compounds in the use for the treatment of drug-resistantfocal seizures, and in the treatment of epilepsy in general.

The present invention demonstrates antiseizure activity of certainisoquinoline alkaloids, and gives another example of the translation ofresults from zebrafish larvae to mice.

The present invention accordingly relates to the screening of otherisoquinoline alkaloids and modified versions thereof for compounds whichare suitable for the prevention and treatment of seizures.

The invention relates to compounds for use in the treatment orprevention of seizures and/or epilepsy.

The invention relates to compounds for use in the treatment orprevention of epilepsy, epileptic seizures and other seizures, and inthe treatment or prevention of the symptoms of epilepsy.

The invention is summarized in the following statements:

A compound comprising a moiety selected from the gr formula oupconsisting of the moieties with formula 1, formula 2, formula 3, formula4, formula 5 and formula 6.

1. An isoquinoline or 1H-2-Benzopyran, selected from the groupconsisting of TMC-120A, TMC-120B, TMC-120C, penicisochroman G,ustusorane B, compound 6 and compound 7,

for use in the treatment or prevention of epilepsy.

2. An isoquinoline or 1H-2-Benzopyran in accordance with statement 1, incombination with halimide or plinabulin,

for use in the treatment or prevention of epilepsy.

3. A method for identifying pharmaceutical compounds against epilepsy,the method comprising the steps of:

-   -   providing a compound which comprises a moiety of a benzene ring        attached to a pyridine ring or puran ring and further comprising        a modified or unmodified furan group attached to the benzene        ring,

and,

as depicted in formula 1 or formula 2

-   -   testing the compound for antiseizure activity.

4. The method according to statement 3, wherein said pyridine or puranring is further methylated as depicted as depicted in Formula 3 orFormula 4

5. The method according to statement 3 or 4, wherein the compoundcomprises the moiety as depicted in Formula 5 or Formula 6.

Thus the methods of the present invention envisage the testing of acompound which comprises a moiety as depicted in formula 1, 2, 3, 4, 5,or 6.

6. The method according to statement 3, 4 or 5, which comprises anisoquinoline or 1H-2-Benzopyran moiety and testing the compound forantiseizure activity.

7. The method according to any one of statement 3 to 6, wherein thecompound with a isoquinoline or 1H-2-Benzopyran moiety is a TMC-120A,TMC-120B, TMC-120C, penicisochroman G, ustusorane B, compound 6 andcompound 7.

8. The method according to any one of statements 3 to 7, whereinhalimide or plinabulin added to said compound for testing antiseizureactivity.

9. The method according to any one of statements 3 to 8, wherein theisoquinoline or 1H-2-Benzypuran is a compound as depicted in FIGS. 1and/or 2, with modified molecular structure or stereochemistry.

10. The method according to any one of statements 3 to 9, whereinanti-seizure activity is determined in a zebrafish model.

11. The method according to any one of statements 3 to 10, whereinanti-seizure activity is further determined in a mammalian model.

12. The method according to any one of statements 3 to 11, furthercomprising the step of testing the compound for a side effect.

13. The method according to any one of statements 3 to 12, furthercomprising the step of formulating a compound with determinedanti-seizure activity into a pharmaceutical composition with anacceptable carrier, for use in the treatment of epilepsy.

DETAILED DESCRIPTION OF THE INVENTION Brief Description of the Figures

FIG. 1. TMC-120A and TMC-120B, chemical structures isolated fromextracts of the fungus Aspergillus insuetus.

FIG. 2. Chemical structures identified based on structural analogy withTMC-120A and TMC-120B. compound 6 stands for7-methylfuro[3,2-h]isoquinoline-3(2H)-one; compound 7 stands for2-(7-methyl-2,3-dihydrofuro[3,2-h]isoquinoline-2-yl)-propan-2-ol.

FIG. 3. Antiseizure hit SK0107. (A) Aspergillus insuetus IBT 28443cultivated on Czapek Yeast extract agar (CYA) and Yeast extract sucroseagar (YES) media for 9 days at 25° C. in the dark. Base peakchromatograms of the crude extract and bioactive fraction SK0107 inpositive electrospray ionization mode. (B, C) Antiseizure activity ofSK0107 in the zebrafish pentylenetetrazole (PTZ) seizure model after 2hours of incubation. PTZ-induced seizure-like behavior is expressed asmean actinteg units per 5 minutes (±SEM) during the 30 minutes recordingperiod (B) and over consecutive time intervals (C). Means are pooledfrom three independent experiments with each 12 replicate wells percondition. Statistical analysis: (B) one-way ANOVA with Dunnett'smultiple comparison test, (C) two-way ANOVA with Bonferroni posttests(GraphPad Prism 5). Significance levels: * p≤05; ** p≤01; *** p≤001.Abbreviation: vehicle, VHC.

FIG. 4. Bioactivity-guided identification of the active compounds ofantiseizure hit SK0107. (A) Aspergillus insuetus IBT 28443 cultivated onCYA media for 9 days in the dark at 25° C. Base peak chromatogram (BPC)of the most bioactive fraction (SK1312) from first reversed phasefractionation in positive electrospray ionization mode. BPCchromatograms of the two most bioactive fractions (SK1414 and SK1415)from the second reversed phase fractionation in positive electrosprayionization mode. UV and HRMS spectra for TMC-120A (II) and UV and HRMSspectra for TMC-120B (III). (B-D) Antiseizure activity of SK1312(n=23-24 replicate wells per condition) (B), SK1414 (n=10-11 replicatewells per condition) (C), and SK1415 (n=22 replicate wells percondition) (D) in the zebrafish pentylenetetrazole (PTZ) seizure modelafter 2 hours of incubation at their maximum tolerated concentration(MTC), MTC/2, and MTC/4. PTZ-induced seizure-like behavior is expressedas mean actinteg units per 5 minutes (±SEM) during the 30 minutesrecording period. (B, D) Data are pooled from two independentexperiments with each 11-12 replicate wells per condition. (B-D)Statistical analysis: one-way ANOVA with Dunnett's multiple comparisontest for comparison of sample+PTZ groups with vehicle (VHC)+PTZ controlgroup, Kruskal-Wallis test with Dunn's multiple comparison test forcomparison of sample+VHC groups with VHC+VHC control group (GraphPadPrism 5). Significance levels: * p≤05; ** p≤01; *** p≤001.

FIG. 5. Behavioral antiseizure analysis of TMC-120A and TMC-120B in thezebrafish PTZ seizure model. Antiseizure activity of TMC-120A (A, B),and TMC-120B (C, D) in the zebrafish pentylenetetrazole (PTZ) seizuremodel after 2 hours of incubation, respectively. PTZ-inducedseizure-like behavior is expressed as mean actinteg units per 5 minutes(±SEM) during the 30 minutes recording period (A, C) and overconsecutive time intervals (B, D). Means are pooled from threeindependent experiments with each 10-12 replicate wells per vehicle(VHC)+PTZ and compound+PTZ condition, and 6-12 replicate wells perVHC+VHC and compound+VHC condition. Statistical analysis: (A, C) one-wayANOVA with Dunnett's multiple comparison test, (B, D) two-way ANOVA withBonferroni posttests (GraphPad Prism 5). Significance levels: * p≤05; **p≤01; *** p≤001.

FIG. 6. Behavioral antiseizure analysis of TMC-120C, penicisochroman Gand ustusorane B in the zebrafish PTZ seizure model. Antiseizureactivity of TMC-120C, penicisochroman G, and ustusorane B in thezebrafish pentylenetetrazole (PTZ) seizure model after 2 hours ofincubation. PTZ-induced seizure-like behavior is expressed as meanactinteg units per 5 minutes (±SEM) during the 30 minutes recordingperiod (A) and over consecutive time intervals (B). Number of replicatewells per condition: 21-22 replicate wells for VHC+PTZ and VHC+VHCconditions, and 8-11 replicate wells for compound+PTZ condition.Statistical analysis: (A) one-way ANOVA with Dunnett's multiplecomparison test, (B) two-way ANOVA with Bonferroni posttests (GraphPadPrism 5). Significance levels: * p≤05; ** p≤01; *** p≤001.

FIG. 7. Electrophysiological antiseizure analysis of TMC-120A andTMC-120B in the zebrafish PTZ seizure model. Noninvasive local fieldpotential recordings from the optic tectum of larvae pre-exposed tovehicle (VHC) and pentylenetetrazole (PTZ), VHC only, compound and PTZ,or compound and VHC. Larvae were incubated with 20 μg/mL TMC-120A orTMC-120B for 2 hours, conform with the maximum tolerated concentrationsand incubation time used in the behavioral assay. Epileptiformdischarges are quantified by the number (mean±SD) (B) and cumulativeduration (mean±SD) (C) of events per 10 minute recording. Larvae areconsidered to possess epileptiform brain activity when three or moreevents occurred during a 10 minute recording (A). Number of replicatesper condition: 19 larvae were used for VHC+PTZ controls, 16 larvae wereused for VHC+VHC controls, 13-14 larvae were used for compound+PTZconditions, and 12 larvae were used for compound+VHC conditions.Statistical analysis: (A) Fisher's exact test with Bonferroni posttest,(B, C) Kruskal-Wallis test with Dunn's multiple comparison test(GraphPad Prism 5). Significance levels: * p≤05; ** p≤01; *** p≤001.

FIG. 8: Antiseizure activity analysis of TMC-120A and TMC-120B in themouse 6-Hz psychomotor seizure model. Drug-resistant psychomotorseizures were induced by electrical stimulation (6 Hz, 0.2 msrectangular pulse width, 3 s duration, 44 mA) through the cornea, 30minutes after i.p. injection of vehicle (VHC, n=20), positive controlvalproate (n=12), TMC-120A (n=4-8), or TMC-120B (n=5-6). Mean seizuredurations (±SD) are depicted. Statistical analysis: one-way ANOVA withDunnett's multiple comparison test (GraphPad Prism 5). Significancelevels: * p≤05; ** p≤01; *** p≤001.

ABBREVIATIONS USED IN THE APPLICATION

ASD, antiseizure drug; CV, column volume; CYA, Czapek Yeast extractagar; dpf, days post-fertilization; DAD, diode array detection; DCM,dichloromethane; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; FA,formic acid; FP7, Seventh Framework Programme; LFP, local fieldpotential; MeCN, acetonitrile; MeOH, methanol; min, minute; MTC, maximumtolerated concentration; NP, natural product; PEG200, polyethyleneglycol M.W. 200; PMR, photomotor response; PTZ, pentylenetetrazole;t_(1/2), half-life; UHPLC-DAD-QTOFMS, Ultra-high performance liquidchromatography-diode array detection-quadrupole time of flight massspectrometry; VHC, vehicle; YES, Yeast extract sucrose agar

Definitions

“Isoquinoline” is a heterocyclic organic compound. It is a structuralisomer of quinolone. Isoquinoline and quinolone are benzopyridines,which are composed of a benzene ring fused to a pyridine ring. Thecompounds of the present invention are isoquinolines which aresubstituted with molecular structures with an unmodified or modifiedfuran group such as dihydrofuran-3-one.

Other compounds of the present invention consist of a benzene ring fusedto a puran ring (1H-2-Benzopyran). The 1H-2-Benzopyran is substitutedwith molecular structures such as dihydrofuran-3-one with an unmodifiedor modified furan group. The above indicated medical use of thecompounds equally comprises the use of the salt form thereof.Pharmaceutically acceptable salts include those described by Berge,Bighley and Monkhouse, J. Pharm. Sci., 1977, 66, 1-19.

Compounds are capable of existing in stereoisomeric forms (e.g.diastereomers and enantiomers) and the invention extends to each ofthese stereoisomeric forms and to mixtures thereof including racemates.The different stereoisomeric forms may be separated one from the otherby the usual methods, or any given isomer may be obtained bystereospecific or asymmetric synthesis. The corresponding stereospecificname and structure have been assigned to the final product where theenantiomeric excess of said product is greater than 70%. Assignment ofabsolute stereochemistry is based on the known chirality of the startingmaterial. In examples where the composition of the final product has notbeen characterised by chiral HPLC, the stereochemistry of the finalproduct has not been indicated. However, the chirality of the maincomponent of the product mixture will be expected to reflect that of thestarting material and the enantiomeric excess will depend on thesynthetic method used and is likely to be similar to that measured foran analogous example (where such an example exists). Thus compoundsshown in one chiral form are expected to be able to be prepared in thealternative chiral form using the appropriate starting material.Alternatively, if racemic starting materials are used, it would beexpected that a racemic product would be produced and the singleenantiomers could be separated by the usual methods. The invention alsoextends to any tautomeric forms and mixtures thereof.

“Seizure” refers to a brief episode of signs or symptoms due to abnormalexcessive or synchronous neuronal activity in the brain. The outwardeffect can vary from uncontrolled jerking movement (tonic-clonicseizure) to as subtle as a momentary loss of awareness (absenceseizure).

Seizure types are typically classified on observation (clinical and EEG)rather than the underlying pathophysiology or anatomy.

I Focal seizures (Older term: partial seizures)

-   -   IA Simple partial seizures—consciousness is not impaired        -   IA1 With motor signs        -   IA2 With sensory symptoms        -   IA3 With autonomic symptoms or signs        -   IA4 With psychic symptoms    -   IB Complex partial seizures—consciousness is impaired (Older        terms: temporal lobe or psychomotor seizures)        -   IB1 Simple partial onset, followed by impairment of            consciousness        -   IB2 With impairment of consciousness at onset    -   IC Partial seizures evolving to secondarily generalized seizures        -   IC1 Simple partial seizures evolving to generalized seizures        -   IC2 Complex partial seizures evolving to generalized            seizures        -   IC3 Simple partial seizures evolving to complex partial            seizures evolving to generalized seizures

II Generalized seizures

-   -   IIA Absence seizures (Older term: petit mal)        -   IIA1 Typical absence seizures        -   IIA2 Atypical absence seizures    -   IIB Myoclonic seizures    -   IIC Clonic seizures    -   IID Tonic seizures,    -   IIE Tonic-clonic seizures (Older term: grand mal)    -   IIF Atonic seizures

III Unclassified epileptic seizures

“Epilepsy” is a condition of the brain marked by a susceptibility torecurrent seizures. There are numerous causes of epilepsy including, butnot limited to birth trauma, perinatal infection, anoxia, infectiousdiseases, ingestion of toxins, tumors of the brain, inherited disordersor degenerative disease, head injury or trauma, metabolic disorders,cerebrovascular accident and alcohol withdrawal.

A large number of subtypes of epilepsy have been characterized andcategorized. The classification and categorization system, widelyaccepted in the art, is that adopted by the International League AgainstEpilepsy's (“ILAE”) Commission on Classification and Terminology [Seee.g., Berg et al. (2010) Epilepsia 51, 676-685:

I. Electrochemical syndromes (arranged by age of onset):

-   -   I.A. Neonatal period: Benign familial neonatal epilepsy (BFNE),        Early myoclonic encephalopathy (EME); Ohtahara syndrome    -   I.B. Infancy: Epilepsy of infancy with migrating focal seizures;        West syndrome; Myoclonic epilepsy in infancy (MEI); Benign        infantile epilepsy; Benign familial infantile epilepsy; Dravet        syndrome; Myoclonic encephalopathy in non-progressive disorders    -   I.C. Childhood: Febrile seizures plus (FS+) (can start in        infancy); Panayiotopoulos syndrome; Epilepsy with myoclonic        atonic (previously astatic) seizures; Benign epilepsy with        centrotemporal spikes (BECTS); Autosomal-dominant nocturnal        frontal lobe epilepsy (ADNFLE); Late onset childhood occipital        epilepsy (Gastaut type); Epilepsy with myoclonic absences;        Lennox-Gastaut syndrome; Epileptic encephalopathy with        continuous spike-and-wave during sleep (CSWS), also known as        Electrical Status Epilepticus during Slow Sleep (ESES);        Landau-Kleffner syndrome (LKS); Childhood absence epilepsy (CAE)    -   I.D. Adolescence-Adult: Juvenile absence epilepsy (JAE);        Juvenile myoclonic epilepsy (JME); Epilepsy with generalized        tonic-clonic seizures alone; Progressive myoclonus epilepsies        (PME); Autosomal dominant epilepsy with auditory features        (ADEAF); Other familial temporal lobe epilepsies    -   I.E. Less specific age relationship: Familial focal epilepsy        with variable foci (childhood to adult); Reflex epilepsies

II. Distinctive constellations

-   -   II.A. Mesial temporal lobe epilepsy with hippocampal sclerosis        (MTLE with    -   II.B. Rasmussen syndrome    -   II.C. Gelastic seizures with hypothalamic hamartoma    -   II.D. Hemiconvulsion-hemiplegia-epilepsy    -   II.E. Epilepsies that do not fit into any of these diagnostic        categories, Distinguished on the basis of Presumed cause        (presence or absence of a known structural or metabolic        condition) or on the basis of Primary mode of seizure onset        (generalized vs. Focal)

III. Epilepsies attributed to and organized by structural-metaboliccauses

-   -   III.A. Malformations of cortical development        (hemimegalencephaly, heterotopias, etc.)    -   III.B. Neurocutaneous syndromes (tuberous sclerosis complex,        Sturge-Weber, etc.)    -   III.C. Tumor    -   III.D. Infection    -   III.E. Trauma

IV. Angioma

-   -   IV.A. Perinatal insults    -   IV.B. Stroke    -   IV.C. Other causes

V. Epilepsies of unknown cause

VI. Conditions with epileptic seizures not traditionally diagnosed asforms of epilepsy per se

-   -   VI.A. Benign neonatal seizures (BNS)    -   VI.B. Febrile seizures (FS)

A newer classification is found in Scheffer et al. (2017) Epilepsia 58,512-521.

A first aspect of the present invention relates to an isoquinolinecompound or a 1H-2-benzopyran compound for use in the treatment orprevention of epilepsy, more particularly for preventing a alleviatingseizures.

Preferred compounds are TMC-120A, TMC-120B, TMC-120C, penicisochroman Gand ustusorane B (depicted in FIG. 1 or 2).

The examples of the present invention used specific compounds isolatedfrom two Aspergillus strains. Herein some are effective in the zebrafishand mouse seizure models, while other show no pharmaceutical activity.The screening did not include all isoquinoline compounds or all1H-2-Benzopyran compounds described in the literature, let alonechemically modified versions.

The present invention relates in another aspect to methods to identifyother isoquinoline or 1H-2-Benzopyran models in the zebrafish and mousemodel to identify compounds with a similar or higher activity thanTMC-120A, TMC-120B, TMC-120C, penicisochroman G, ustusorane B, and orwith better ADMET properties.

Another aspect of the present invention relates to methods foridentifying compounds which are active in models for determininganti-seizure activity. Envisaged compounds are structurally related tothe compounds depicted in FIG. 1. The envisaged compounds all comprise amoiety of a benzene ring fused to a pyridine ring or a moiety of abenzene ring fused to a puran ring. The envisaged compounds typicallyfurther comprise a modified or unmodified furan group attached to thebenzene ring.

Thus the compounds comprise a benzene ring fused to a) a pyridine ringor a puran ring and b) fused to a modified or unmodified furan group,The envisaged compounds are typically further methylated on the pyridineor puran group, generally on the position as shown in FIG. 1.

Examples of suitable compounds are e.g. disclosed in Kuramochi andTsubaki (2015) Nat. Prod. 78, 1056-1066, Kuramochi et al. (2013) J NatProd. 76, 1737-1745, Bumbamrung et al. (2014) Phytochem Lett. 10, 13-18,and Slack et al (2009) Mycol. Res. 113, 480-490

Methods of the present invention for drug screening can beadvantageously performed with a zebrafish model which is suitable forlarge-scale screening and captures the complexity of a whole bodyorganism and the central nervous system. As a vertebrate, zebrafish arehighly similar to humans due to a high genetic, physiological andpharmacological conservation. Moreover, given the small size of embryosand larvae, they fit in the well of microtiter plates and hence aresuitable for medium to high-throughput testing [Howe et al, MacRae andPeterson, Khan et al. cited above]. Given the low volumes used in 96-and 384-well plates, zebrafish larvae only require small amounts ofsample in the low microgram range when added to their swimming water andeven less when administered by injection. This property is of particularinterest for marine natural product drug discovery, where material isoften scarce [West and Crawford, cited above]. A particular suitablelarval zebrafish seizure and epilepsy model, is the larval zebrafishpentylenetetrazole (PTZ) seizure model. This model has the followingadvantages 1) the model has been extensively characterized in terms ofbehavioral and non-behavioral seizure markers, 2) it has beenpharmacologically characterized with ASDs on the market, 3) resultstranslate well to rodent models, 4) seizures can easily and rapidly beinduced by a single administration of the convulsant drug to the larva'saqueous environment, and 5) seizures can be quantified automatically byvideo recording [Baraban et al. (2005) Neuroscience 131, 759-768;Afrikanova et al. (2013) PLoS One 8, e54166; Berghman et al. (2007)Epilepsy Res 75, 18-28; Buenafe et al. (2013) ACS Chem Neurosci 4,1479-1487; Patel and Patel (2011) J. App. Pharm. Sci. 1, 167-171].

Within the framework of PharmaSea, two isoquinoline alkaloids TMC-120Aand TMC-120B were isolated from the bioactive marine-derived fungusAspergillus insuetus, which was isolated from a seawater trap set in theNorth Sea, in between Norway and Denmark. Both compounds wereinvestigated for antiseizure activity in the larval zebrafish PTZseizure model, after acute and chronic exposure. In addition,electrophysiological analysis from the zebrafish midbrain demonstratedthat TMC-120A and TMC-120B also significantly lowered PTZ-inducedepileptiform discharges. In addition, other isoquinoline compounds wereidentified based on structural homology to TMC-120A and TMC-120B. Thesecompounds are TMC-120C, penicisochroman G, ustusorane B, compound 6 andcompound 7. All compounds demonstrated to have antiseizure activity inthe larval zebrafish PTZ seizure model, after acute exposure.

Methods of the present invention for drug screening can be equallyperformed in a mammalian model, wherein generally those compounds aretested which already gave positive results in the above mentionedzebrafish model.

Accordingly, TMC-120A and TMC-120B were tested in the mouse 6-Hz (44 mA)psychomotor seizure model.

Herein, treatment with TMC-120A and TMC-120B shortened the seizureduration, thereby confirming the antiseizure activity observed in thezebrafish model. These results not only confirmed the translation ofresults from zebrafish larvae to mice but also indicates that TMC-120Aand TMC-120B are effective against drug-resistant focal seizures.

Methods of the present invention for drug screening can further comprisethe step of determining parameters such as absorption, distribution,metabolism, and excretion—toxicity.

In summary, based on the prominent antiseizure activity seen in astandard zebrafish and mouse seizure model and their ADMETcharacteristics, the present invention claims TMC-120A, TMC-120B,TMC-120C, penicisochroman G, ustusorane B, compound 6 and compound 7 ascompounds for use in the treatment of seizures and/or epilepsy.

EXAMPLES Example 1. Methods

1.1. Chemical Experimental Procedures

Ultra-high performance liquid chromatography-diode arraydetection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS)was performed on an Agilent Infinity 1290 UHPLC system (AgilentTechnologies, Santa Clara, Calif., USA) equipped with a diode arraydetector (DAD). Separation was achieved on an Agilent Poroshell 120phenyl-hexyl column (2.1×150 mm, 2.7 μm) with a flow of 0.35 mL/min at60° C. using a linear gradient 10% acetonitrile (MeCN) in Milli-Q waterbuffered with 20 mM formic acid (FA) increased to 100% in 15 min stayingthere for 2 min, returned to 10% in 0.1 min and kept there for 3 minbefore the following run. MeCN was LC-MS grade. MS detection was done onan Agilent 6550 iFunnel QTOF MS equipped with Agilent Dual Jet Streamelectrospray ion source with the drying gas temperature of 160° C. andgas flow of 13 L/min and sheath gas temperature of 300° C. and flow of16 L/min. Capillary voltage was set to 4000 V and nozzle voltage to 500V. Data processing was performed using Agilent MassHunter QualitativeAnalysis for quadrupole time of flight (version B.07.00).Pre-fractionation was performed using flash chromatography of the crudeextract with an Isolera one automated flash system (Biotage, Uppsala,Sweden). Purification of compounds was conducted using a Waters 600Controller (Milford, Mass., USA) coupled to a Waters 996 PhotodiodeArray Detector. One and two dimensional (1D and 2D) NMR experiments wereacquired using standard pulse sequences on either a 600 MHz BrukerAscend spectrometer with a SmartProbe (BBO), a 400 MHz Bruker Ascendspectrometer with a Prodigy cryoprobe or a 800 MHz Bruker Avancespectrometer with a 5 mm TCI cryoprobe. Optical rotations were measuredon a Perkin Elmer 341 polarimeter (Perkin Elmer, Waltham, Mass., USA).

1.2. Microbial Strain and Microbial Cultivation

Aspergillus insuetus IBT 28443 and IBT 28485 were from the IBT culturecollection at the Department of Biotechnology and Biomedicine, TechnicalUniversity of Denmark. The fungus Aspergillus insuetus IBT 28443 wascollected at the Galathea 3 expedition and isolated from a seawater trapset in the North Sea, in between Denmark and Norway.

Aspergillus insuetus IBT 28443 was cultivated on one CYA and one YESmedia plates for 9 days in the dark at 25° C. for the original combinedsmall scale extract. For the individual small scale extracts the funguswas cultivated on eight plates of CYA, eight plates of YES and eightplates of OAT for 9 days in the dark at 25° C. For the large scaleextract the fungus was cultivated on 250 plates of CYA for 9 days in thedark at 25° C.

Aspergillus insuetus IBT 28485 was cultivated on 220 CYA plates for 7days in the dark at 25° C.

1.3. Microbial Extraction and Isolation

For the original combined small scale extract of Aspergillus insuetusIBT 28443 the two plates in total (one CYA and one YES) were extractedwith 40 mL ethyl acetate (EtOAc) containing 1% FA. The crude extract wasthen fractionated on a reversed phase C₁₈ flash column (Sepra ZT,Isolute, 10 g) using an Isolera One automated flash system (Biotage,Uppsala, Sweden). The gradient used was 15%-100% MeCN buffered with 20mM FA over 28 min (12 mL/min). Six flash fractions were automaticallycollected based on UV signal (210 nm and 254 nm). For the individualsmall scale extracts on CYA, YES and OAT each of the separate set ofeight plates were extracted with 150 mL EtOAc with 1% FA and for thelarge scale extract on CYA it was extracted with 150 mL EtOAc with 1% FAfor every 10 plates. All the crude extracts were fractionated on areversed phase C₁₈ flash column (Sepra ZT, Isolute, 25 g/33 mL) usingthe Isolera One automated flash system. The gradient was 10% stepwise(12 column volumes) from 15% to 100% MeCN buffered with 20 mM FA with aflow of 25 mL/min. Fractions were collected manually for every 10%. Forthe large scale extract the most bioactive fraction (25% MeCN) wasfractionated on a reversed phase Isolute SPE column (500 mg/3 mL) usingmethanol (MeOH) buffered with 20 mM FA. The compounds were eluted with 2column volumes (CV) per fraction: 15% MeOH, 20% MeOH, 30% MeOH, 40%MeOH, 50% MeOH, 60% MeOH, 80% MeOH and 100% MeOH. From the 60% MeOH and80% MeOH isolera fractions TMC-120A and TMC-120B separation was achievedon a Gemini C₆ Phenyl, 5 μm, 250×10 mm column (Phenomenex, Torrance,Calif., USA) with a flow of 4 mL/min. A linear gradient was used of 40%MeCN in Milli-Q water with 20 mM FA going to 70% MeCN in 30 min.

For the large scale extract of Aspergillus insuetus IBT 28485, the 220Petri dish plates were extracted with 150 mL EtOAc with 1% FA for every10 plates. The crude extract was fractionated on a diol flash column(Diol, 25 g, 33 mL) using the Isolera One automated flash system. Thecompounds were eluted with 2 CV per fraction: heptane, heptane 1:1dichloromethane (DCM), DCM, DCM 1:1 EtOAc, EtOAc, EtOAc 1:1 MeOH andMeOH. The DCM, DCM 1:1 EtOAc and EtOAc fractions were furtherfractionated on the Isolera One flash system on a reversed phase C₁₈flash column (15 μm/100 Å, 10 g/15 mL) using MeOH buffered with 20 mM FAwith a flow of 15 mL/min. Compounds were eluted with 6 CV per fraction:35% MeOH, 40% MeOH, 42% MeOH, 45% MeOH, 47% MeOH, 50% MeOH, 55% MeOH,60% MeOH, 70% MeOH, 80% MeOH and 100% MeOH. From the 42% and 45% MeOHisolera fractions TMC-120A and TMC-120B separation was achieved on aGemini C₆ Phenyl, 5 μm, 250×10 mm column (Phenomenex, Torrance, Calif.,USA) with a flow of 4 mL/min using a linear gradient 40% MeCN in Milli-Qwater with 20 mM FA going to 70% MeCN in 30 min. TMC-120C (from fraction40% MeOH) separation was achieved on a Gemini C₆ Phenyl, 5 μm, 250×10 mmcolumn (Phenomenex, Torrance, Calif., USA) with a flow of 4 mL/min andusing a linear gradient of 30% MeCN in Milli-Q water going to 60% MeCNin 30 min. Penicisochroman G and ustusorane B (from fractions 70% MeOHand 80% MeOH) separation was achieved on a Gemini C₆ Phenyl, 5 μm,250×10 mm column (Phenomenex, Torrance, Calif., USA) with a flow of 4ml/min using a linear gradient 65% MeCN in Milli-Q water with 20 mM FAgoing to 85% MeCN in 30 min.

TMC-120A: pale yellow solid; [α]_(D) ²⁰ −5 (c 0.51, MeOH); UV (MeCN)Amax: 214 nm, 245 nm, 344 nm, 360 nm; HRESIMS m/z 242.1177[M+H]+(calculated for C₁₅H₁₆NO₂, m/z 242.1176, Δ −0.32)

TMC-120B: slightly pale yellow needles; UV (MeCN) Amax: 214 nm, 240 nm,262 sh nm, 275 nm, 296 sh nm, 306 nm, 369 nm; HRESIMS m/z 240.1019[M+H]+(calculated for C₁₅H₁₄NO₂, m/z 240.1019, Δ 0.14)

1.4. Compound and Sample Preparation

For experiments with zebrafish larvae, dry samples and compounds weredissolved in 100% dimethyl sulfoxide (DMSO, spectroscopy grade) as100-fold concentrated stocks and diluted in embryo medium to a finalconcentration of 1% DMSO content, except for PTZ which was dissolved inembryo medium (0% DMSO). Control groups were treated with 1% DMSO (VHC)in accordance with the final solvent concentration of tested samples orcompounds. For mice experiments, a mixture of poly-ethylene glycol M.W.200 (PEG200) and 100% DMSO (spectroscopy grade) (1:1 PEG200:DMSO) wasused as solvent and VHC. Pentylenetetrazole and valproate were purchasedfrom Sigma-Aldrich.

1.5. Experimental Animals

All animal experiments carried out were approved by the Ethics Committeeof the University of Leuven (approval numbers 101/2010, 061/2013,150/2015, 023/2017, and 027/2017) and by the Belgian Federal Departmentof Public Health, Food Safety & Environment (approval number LA1210199).

Zebrafish

Adult zebrafish (Danio rerio) stocks of AB strain (ZebrafishInternational Resource Center, Oregon, USA) were maintained at 28.0° C.,on a 14/10 hour light/dark cycle under standard aquaculture conditions.Fertilized eggs were collected via natural spawning and raised in embryomedium (1.5 mM HEPES, pH 7.2, 17.4 mM NaCl, 0.21 mM KCl, 0.12 mM MgSO4,0.18 mM Ca(NO3)2, and 0.6 μM methylene blue) at 28.0° C., under constantlight with regards to the zebrafish PTZ seizure model and under a 14/10hour light/dark cycle with regards to the zebrafish photomotor responseassay

Mice

Male NMRI mice (weight 18-20 g) were acquired from Charles RiverLaboratories and housed in poly-acrylic cages under a 14/10-hourlight/dark cycle at 21° C. The animals were fed a pellet diet and waterad libitum, and were allowed to acclimate for one week beforeexperimental procedures were conducted. Prior to the experiment, micewere isolated in a poly-acrylic cage with a pellet diet and water adlibitum for habituation overnight in the experimental room, to minimizestress.

1.6 Zebrafish Photomotor Response Assay

Behavioral Analysis

Experiments were performed as described in Copmans et al. (2016) J.Biomol Screen 21, 427-436. In the primary screen one replicate well wasused per sample tested and each experimental plate contained 6 internalcontrol wells. Each well held 5 embryos that were incubated with samplefor 2 hours prior to behavioral recording at 32 hpf. A neuroactive hitwas defined as a marine NP that modified the PMR such that itsbehavioral fingerprint (16 pseudo Z-scores that together describe theembryonic motion over a 30 second recording period) contained at leastone pseudo Z-score with an absolute value equal to or exceeding 5.

Toxicity Evaluation

Each behavioral analysis was followed by visual evaluation of theembryos under a light microscope to assess toxicity of treatment.Overall morphology, heartbeat, and touch response were investigated.Marine NPs were scored normal or toxic. When embryos showed normalmorphology, normal or lowered heartbeat, and normal or lowered touchresponse the treatment was considered to be normal. In case of anabnormal morphology and/or absence of touch response or heartbeat atreatment was considered to be toxic.

1.7. Zebrafish Pentylenetetrazole Seizure Model

Toxicity Evaluation

Maximum tolerated concentration (MTC) was determined prior to furtherexperiments and used as the highest test concentration. Experiments weredescribed as described in Copmans et al. (2018) ACS chemicalneuroscience. MTC was investigated by exposing 12 larvae of 6 dpf to arange of concentrations in a 100 μl volume during 18 hours. Thefollowing parameters were investigated after 2 and 18 hours of exposure:touch response, morphology, posture, edema, signs of necrosis, swimbladder, and heartbeat. MTC was defined as the highest concentration atwhich no larvae died nor showed signs of toxicity or locomotorimpairment in comparison to VHC-treated control larvae.

For screening purposes, no MTC was determined, but behavioral analysiswas followed by visual evaluation of the larvae under a light microscopeto assess toxicity of treatment. Overall morphology, heartbeat, andtouch response were investigated. Marine NPs were scored normal ortoxic. When embryos showed normal morphology, heartbeat, and touchresponse the treatment was considered to be normal. In case of anabnormal morphology and/or absence of touch response or heartbeat atreatment was considered to be toxic.

Behavioral Analysis

Experiments were performed as [Add references please:

Copmans (2018), ACS chemical neuroscience; Copmans et al. (2018)Neurochem. Int. 112, 124-133; Afrikanova et al. (2013) PLoS One 8,e54166; Orellana-Paucar et al. (2012) Epilepsy Behav 24, 14-22]. Inbrief, a single 7 dpf larva was placed in each well of a 96-well plateand treated with either VHC (1% DMSO) or test compound in a 100 μlvolume. Larvae were incubated in dark for 2 hours at 28° C., whereafter100 μl of either VHC (embryo medium) or 40 mM PTZ (20 mM workingconcentration) was added to each well. Next, within 5 minutes the96-well plate was placed in an automated tracking device (ZebraBoxViewpoint, France) and larval behavior was video recorded for 30minutes. The complete procedure was performed in dark conditions usinginfrared light. Total locomotor activity was recorded by ZebraLabsoftware (Viewpoint, France) and expressed in actinteg units, which isthe sum of pixel changes detected during the defined time interval (5minutes). Larval behavior was depicted as mean actinteg units per 5minutes in the 30 minute recording period and over consecutive timeintervals. Data are expressed as mean±SD for single experiments withregards to screening, and as mean±SEM for single experiments and forindependent experiments of which the means or data are pooled.

In the first secondary screen three replicate wells were used per sample(100 μg/mL) tested and each experimental plate contained 12 internalcontrol wells. In the second secondary screen six replicate wells wereused per sample and concentration tested (100, 33, and 11 μg/mL), again12 internal control wells were used per experimental plate.

1.7. Electrophysiology

Non-invasive LFP recordings were measured from the midbrain (optictectum) of 7 dpf zebrafish larvae pre-incubated with VHC only, PTZ only,compound and VHC, or compound and PTZ. Experiments were performed asdescribed in Copmans et al. (2018) Neurochem. Int. 112, 124-133;

Copmans (2018) ACS chemical neuroscience. In brief, larvae wereincubated for approximately 2 hours with VHC (1% DMSO) or test compound(1% DMSO) in a 100 μL volume. After incubation, an equal volume of VHC(embryo medium) or 40 mM PTZ (20 mM working concentration) was added tothe well for 15 minutes prior to recording. These steps occurred at 28°C., while further manipulation and electrophysiological recordingsoccurred at room temperature (±21° C.). The larva was embedded in 2% lowmelting point agarose (Invitrogen) and the signal electrode (anelectrode inside a soda-glass pipet (1412227, Hilgenberg) pulled with aDMZ Universal Puller (Zeitz, Germany), diameter ±20 microns, containingartificial cerebrospinal fluid (ACSF: 124 mM NaCl, 10 mM glucose, 2 mMKCl, 2 mM MgSO₄, 2 mM CaCl₂), 1.25 mM KH₂PO₄, and 26 mM NaHCO₃, 300-310mOsmols)) was positioned on the skin covering the optic tectum. Adifferential extracellular amplifier (DAGAN, USA) amplified the voltagedifference between the signal (measured by the signal electrode) and thereference electrode. The differential signal was band pass filtered at0.3-300 Hz and digitized at 2 kHz via a PCI-6251 interface (Nationalinstruments, UK) using WinEDR (John Dempster, University of Strathclyde,UK). A grounding electrode grounded the electrical system. Allelectrodes were connected with ACSF. Each recording lasted 600 seconds.Manual analysis was completed by quantification of the number,cumulative duration, and mean duration of epileptiform-like events withClampfit 10.2 software (Molecular Devices Corporation, USA). Anelectrical discharge was considered epileptiform if it was apoly-spiking event comprising at least 3 spikes with a minimum amplitudeof three times the baseline amplitude and a duration of at least 100milliseconds. Data are expressed as mean±SD.

1.8 Mouse 6-Hz Psychomotor Seizure Model

1.8. Mouse 6-Hz Psychomotor Seizure Model

Experiments were performed as described in Copmans et al. (2018) ACSchemical neuroscience. In brief, 50 μL (injection volume was adjusted tothe individual weight) of VHC (PEG200:DMSO 1:1) or treatment (an ASD ortest compound dissolved in VHC) was i.p. injected in NMRI mice (averageweight 32 g, range 28-36 g) and after 30 minutes psychomotor seizureswere induced by low frequency, long duration corneal electricalstimulation (6 Hz, 0.2 ms rectangular pulse width, 3 s duration, 44 mA)using an ECT Unit 5780 (Ugo Basile, Comerio, Italy). Mice were manuallyrestrained and a drop of ocular anesthetic (0.5% lidocaine) was appliedto the corneas before stimulation. Following electrical currentstimulation, the mouse was released in a transparent cage for behavioralobservation, which was video-recorded. VHC-treated mice typicallydisplayed stun, twitching of the vibrissae, forelimb clonus, and Straubtail. In addition, facial and mouth jerking as well as head nodding wereobserved occasionally. Seizure durations were measured during theexperiment by experienced researchers, familiar with the differentseizure behaviors. In addition, seizure durations were determined byblinded video analysis to confirm or correct the initial observations.Data are expressed as mean±SD.

Example 2. Zebrafish-Based Antiseizure Drug Discovery

2009 marine NPs, i.e., extracts and pre-fractionated fractions, providedby the different PharmaSea partners, were screened for neuroactivity ata concentration of 100 μg/mL (2 hours incubation time) using thezebrafish PMR assay. The PMR was described by a behavioral fingerprintof 16 pseudo Z-scores that represent the embryonic motion over a 30second recording period using the first and third quantile (Q1 and Q3)for each of the 8 time periods. A neuroactive hit was defined as amarine NP that modified the PMR such that its behavioral fingerprintcontained at least one pseudo Z-score with an absolute value equal to orexceeding 5. Each PMR-assay was followed by visual evaluation of theembryos under a light microscope to assess toxicity of treatment. Only109 marine NPs were observed to cause toxicity. All other treatments didnot induce toxicity under the test conditions, whereof 332 wereneuroactive and 1568 samples were inactive. The 332 neuroactive hitsunderwent antiseizure analysis at a concentration of 100 μg/mL (2 hoursincubation time) using the zebrafish PTZ seizure model. In this modelthe convulsant PTZ (20 mM) is administered to the swimming water of 7days post-fertilization (dpf) larvae and induces typical seizure-likebehavior that is characterized by high-speed swimming, whirlpool-likecircling, clonus-like seizures, and loss of posture. An antiseizure hitwas defined as a marine NP that significantly lowered the stronglyelevated larval locomotion as a result of PTZ-induced seizures.Initially, 97 antiseizure hits were identified that did not result intoxicity, whereof 43 were confirmed in a second screen using twice thenumber of larvae per sample. Moreover, the latter screen investigatedconcentration-dependent effects by analyzing a three-fold serialdilution from 100 μg/mL onwards. Hit prioritization was based onefficacy, concentration-dependency, and sample availability.

Among prioritized hits was marine NP SK0107, one of the more polarfractions from initial reverse phase chromatographic separation of thecrude extract of Aspergillus insuetus IBT 28443 (FIG. 3A), which wasisolated from a seawater trap set in the North Sea, in between Norwayand Denmark. Aspergillus insuetus is a filamentous fungus belonging toAspergillus section Usti that includes species from soil, foods, andindoor air environments but also from marine isolates. Marine-derivedfungal isolates with Aspergillus species as a common source, have beenseen to yield a plethora of biologically active compounds includingstructurally unique secondary metabolites. Prior to further experimentsthe maximum tolerated concentration (MTC) of SK0107 was determined,which was defined as the highest concentration at which no larvae diednor showed signs of toxicity or locomotor impairment in comparison tovehicle (VHC)-treated control larvae. The MTC was observed to be 50μg/mL and used as the highest test concentration in all subsequenttests. To validate the results obtained during the course of screeningthe antiseizure activity of SK0107 was investigated in the larvalzebrafish PTZ seizure model at the MTC, MTC/2, and MTC/4 (two-foldserial dilution, 2 hours incubation time) in three independentexperiments (FIG. 3B-C). In line with former results, the antiseizurehit SK0107 showed significant concentration-dependent activity againstPTZ-induced seizure behavior, both during the 30 minute (min) recordingperiod (p≤0.001 and p≤01) (FIG. 3B) as within 5 min time intervals(p≤001, p≤01, and p≤05) (FIG. 3C).

Example 3. Bioactivity-Guided Identification of Active Compounds

To identify the active constituents of SK0107 that are responsible forits antiseizure activity bioactivity-guided purification was performedof Aspergillus insuetus IBT 28443. In the crude extract of Aspergillusinsuetus dereplication using ultra-high performance liquidchromatography-diode array detection-quadrupole time of flight massspectrometry (UHPLC-DAD-QTOFMS) tentatively identified an abundantpresence of the sesterterpenoids, ophiobolins (inactive, data notshown). Before any large scale cultivation, small scale extracts wereprepared of the fungus cultivated individually on CYA, YES and OATmedia, as the tested bioactive extract was of the combined cultivationon both CYA and YES media. This was done in hope of finding a mediumwhere the production of ophiobolins was reduced and other compoundspresented in a higher concentration than the original crude extract. CYAmedium was chosen based on the activity of fractions from the crudeextract and based on the reduced concentration of ophiobolins (data notshown).

A large scale extract was prepared from cultivation of Aspergillusinsuetus IBT 28443 on CYA media for 9 days in the dark at 25° C. andbioactivity-guided purification was performed through several reversedphase purification steps until single compound isolation. In the twomost bioactive fractions from the second fractionation of the crudeextract, i.e., SK1414 and SK1415 (FIG. 4C-D), three compounds weretentatively identified by UHPLC-DAD-QTOFMS (FIG. 4A). One compound withthe pseudomolecular ion, [M+H]⁺ m/z 351.1818 (mass accuracy −0.77 ppm)and two related compounds that were seen to coelute by firstfractionation with the pseudomolecular ions, [M+H]⁺ m/z 242.1177 (massaccuracy −0.32 ppm) and m/z 240.1019 (mass accuracy 0.14 ppm). Themolecular formulas was based on the pseudomolecular ions m/z 242.1177and m/z 240.1019 established to be C₁₅H₁₅NO₂ and C₁₅H₁₃NO₂ respectively.A search in Antibase2012 for the formulas revealed the possiblecandidates to be the isoquinoline alkaloids TMC-120A and TMC-120B forthe two related compounds (FIG. 4A). This was supported by UV/vis dataconsistent with litterature, with both compounds displayingcharacteristic UV/vis spectra, and production by related fungal species(Aspergillus ustus). The structure of TMC-120B was confirmed byelucidation with 1D and 2D NMR spectroscopy and comparison of ¹H and ¹³Cchemical shifts to literature data [Kohno et al (1999) Tetrahedron 55,11247-11252]. As only trace amounts of compounds TMC-120A and TMC-120Bcould be purified from the crude extract of Aspergillus insuetus IBT28443, various closely related species belonging to Aspergillus sectionUsti (Table 1) were investigated by HRMS, MS/HRMS and UV/vis data tofind a better fungal producer. Aspergillus insuetus IBT 28485 was chosenbased on its production of TMC-120A and TMC-120B as some of the maincompounds. TMC-120A and TMC-120B were purified in higher amounts (1.0mg), and the structure of TMC-120A was confirmed by 1D and 2D NMRspectroscopy and comparison of ¹H and ¹³C chemical shifts and opticalrotation to literature data [Kohno et al (1999) Tetrahedron 55,11247-11252]. Some of the fungus other major metabolites were isolatedi.e. the structural analogues TMC-120C, penicisochroman G and ustusoraneB with HRMS, UV/Vis and ¹H and ¹³C NMR data consistent with literature[Kohno et al (1999) Tetrahedron 55, 11247-11252, Bunbamrung et al (2014)Phytochem. Lett. 10, 13-18, Lu et al (2009) J. Nat. Prod. 72,1761-1767].

TABLE 1 Potential TMC-120A and TMC-120B producing strains fromAspergillus section Usti. IBT number Species 4133 Aspergillus ustus10619 Aspergillus ustus 28485 Aspergillus insuetus 914826 Aspergilluscalidoustus

Closely related species belonging to Aspergillus section Usti from theIBT culture collection at the Department of Biotechnology andBiomedicine that are potential TMC-120A and TMC-120B producing strains.

Table 2 shows NMR spectroscopic data (400 MHz, CDCl₃, δ in ppm, J in Hz)for TMC-120B isolated from the crude extract of Aspergillus insuetus IBT28443.

TABLE 2 NMR spectroscopic data for TMC-120B TMC-120B Position δ_(H)(mult, J) δ_(C) 1 — — 2 — 145.6 3 — 182.1  3a — 119.3 4 7.81 124.4d(8.6) 5 7.36 120.8 d(8.6)  5a — 141.3 6 7.53 s 119.8 7 — 156.7 8 — — 99.55 s 146.3  9a — 114.6  9b — 164.0 10  2.74 s 24.9 11  — 133.7 12 2.42 s 17.6 13  2.24 s 20.5

Table 3 shows NMR spectroscopic data (800 MHz, CDCl₃, δ in ppm, J in Hz)for TMC-120A and TMC-120B isolated from the crude extract of Aspergillusinsuetus IBT 28485

TABLE 3 NMR spectroscopic data for TMC-120A and TMC-120B TMC-120ATMC-120B Position δ_(H) (mult, J) δ_(C) δ_(H) (mult, J) δ_(C) 1 — — — —2 4.85 d(4.0) 91.8 — 145.8 3 — 199.8 — 182.4  3a — 117.8 — 119.6 4 7.71d(8.6) 124.3 7.80 d(8.6) 124.4 5 7.30 d(8.6) 120.3 7.36 d(8.6) 120.8  5a— 142.5 — 141.5 6 7.53 s 119.9 7.53 s 119.8 7 — 157.4 — 156.8 8 — — — —9 9.55 s 146.6 9.54 s 146.4  9a — 115.2 — 114.8  9b — 174.1 — 164.2 10 2.75 s 24.7 2.74 s 24.9 11  2.46 m 31.3 — 134.0 12  0.92 d(6.9) 15.92.42 s 17.8 13  1.23 d(6.9) 19.0 2.24 s 20.6

Table 4 shows NMR spectroscopic data for penicisochroman G andustusorane B (600 MHz, CDCl₃, δ in ppm, J in Hz) and TMC-120C (800 MHz,CDCl₃, δ in ppm, J in Hz) isolated from the crude extract of Aspergillusinsuetus IBT 28485.

TABLE 4 NMR spectroscopic data for penicisochroman G, ustusorane B andTMC-120C Penicisochroman G Ustusorane B TMC-120C δ_(H) δ_(H) δ_(H)Position (mult, J) δ_(C) (mult, J) δ_(C) (mult, J) δ_(C) 1 — — — — — — 24.39 d(3.9) 90.4 — 144.6 — 110.3 3 — 200.6 — 182.2 — 198.7  3a — 120.0 —120.7 — 115.8 4 7.43 d(8.0) 123.9 7.52 d(8.0) 122.9 7.52 d(8.2) 124.5 56.57 d(8.0) 117.0 6.63 d(8.0) 116.2 6.99 d(8.2) 119.6  5a — 142.0 —139.6 — 142.1 6 5.65 s 101.7 5.67 s 100.7 7.14 s 119.6 7 — 160.0 — 158.5— 156.7 8 — — — — — — 9 5.22 s 62.8 5.26 s 61.5 9.46 s 146.8  9a — 108.9— 107.7 — 114.7  9b — 168.3 — 158.9 — 171.6 10  1.95 s 20.1 1.95 s 18.72.50 s 23.9 11  2.31 m 31.2 — 129.9 2.40 m 34.1 12  0.84 d(6.9) 15.82.33 s 16.2 0.96 16.1 13  1.13 d(6.9) 19.0 2.06 s 19.0 1.21 d(6.6) 15.7

Example 4. TMC-120A and TMC-120B Ameliorate Seizures in the ZebrafishPTZ Seizure Model

To confirm that TMC-120A and TMC-120B isolated from the most bioactivefractions are indeed the active constituents, their antiseizure activitywas investigated in the zebrafish PTZ seizure model (FIG. 5). Larvaewere treated with TMC-120A or TMC-120B for 2 hours, using their MTC,MTC/2, and MTC/4, conform with the conditions used for the crude extractand purified fractions. TMC-120B, but not TMC-120A, significantlylowered PTZ-induced seizure behavior at its MTC in the 30 min recordingperiod (p≤01) (FIGS. 5A and C). A more detailed analysis of the 30 minrecording period into 5 min time intervals revealed a significantreduction of PTZ-induced seizure behavior for both compounds at theirMTCs. More specifically, within the 10-30 min time window with p≤05,p≤01, and p≤001 at different time intervals in case of TMC-120A andwithin the 15-25 min time window with p≤05 in case of TMC-120B (FIGS. 5Band D). No significant antiseizure activity was seen at lowerconcentrations, except for TMC-120A at the MTC/2 (p≤05) in the last 5min time interval. These data demonstrate that TMC-120A and TMC-120Bhave antiseizure activity and confirm that the isolated compounds areindeed active constituents of the antiseizure hit SK0107, and thebioactive fractions SK1312, SK1414, and SK1415. The higher antiseizureefficacy of the bioactive extract and fractions in comparison to theseobserved for the individual compounds is possibly due to a synergisticaction

Example 5. TMC-120C, Penicisochroman G and Ustusorane B AmeliorateSeizures in the Zebrafish PTZ Seizure Model

The antiseizure activity of TMC-120C, penicisochroman G and ustusorane Bwas investigated in the zebrafish pentylenetetrazole (PTZ) seizure modelafter 2 hours of incubation (FIG. 6). All three compounds loweredPTZ-induced seizure behavior in the 30 min recording period at thetested concentration, which was significant for penicisochroman G(p≤001) and ustusorane B (p≤001) (FIG. 6A). A more detailed analysis ofthe 30 min recording period into 5 min time intervals showed asignificant reduction of PTZ-induced seizure behavior for all threecompounds. More specifically, within the 10-30 min time window forTMC-120C (p≤05, p≤01 and p≤001) and over the entire recording period forpenicisochroman G (p≤001) and ustusorane B (p≤01 and p≤001) (FIG. 6B).Hence, TMC-120C, penicisochroman G and ustusorane B show antiseizureactivity.

Example 6. TMC-120A and TMC-120B Ameliorate Epileptiform Brain Activityin the Zebrafish PTZ Seizure Model

To determine whether TMC-120A and TMC-120B besides antiseizure activityalso ameliorate the PTZ-induced hyperexcitable state of the brain thatis characterized by epileptiform discharges, local field potential (LFP)recordings were non-invasively measured from the midbrain (optic tectum)of zebrafish larvae (FIG. 7). Larvae were treated with either VHC ortest compound (MTC and 2 hours incubation time were used in line withprevious experiments) followed by a 15 min during exposure to PTZ or VHCprior to LFP measurements. Pre-exposure to PTZ but not to VHC resultedin a significant increase of epileptiform electrical discharges.Pre-incubation with TMC-120A significantly lowered the percentage oflarvae with PTZ-induced epileptiform activity by more than half (p≤001)(FIG. 7A). A larva was considered to have epileptiform brain activitywhen at least 3 electrical discharges were seen during the 10 minrecording that fulfilled the pre-defined requirements of an epileptiformevent (see methods). In addition, pre-incubation with TMC-120A orTMC-120B significantly lowered the number (p≤001, and p≤01,respectively) and the cumulative duration (p≤001, and p≤05,respectively) of PTZ-induced epileptiform events over the 10 minrecording period (FIGS. 7B and C). Thus, TMC-120A and TMC-120B showanti-epileptiform activity and likely display their antiseizureproperties by counteracting the hyperexcitable state of the brain.

Example 7. TMC-120A and TMC-120B Ameliorates Focal Seizures in the Mouse6-Hz (44 mA) Psychomotor Seizure Model

We investigated whether the antiseizure action of TMC-120A and TMC-120Bobserved in the larval zebrafish model translates to a standard rodentseizure model. From the available rodent seizure models we chose themouse 6-Hz (44 mA) psychomotor seizure model, a gold standard in currentASD discovery efforts that is useful for screening and can detectcompounds with novel antiseizure mechanisms and with potential againstdrug-resistant seizures. Seizures are characterized by a minimal clonicphase and stereotype automatistic behaviors, typically seen as stun,forelimb clonus, Straub tail, and twitching of the vibrissae [Barton etal. (2001) Epilepsy Res 47, 217-227; Buenafe et al. (2013) ACS ChemNeurosci 4, 1479-1487.]. Male NMRI mice were intraperitoneally (i.p.)injected with a 50 μL volume (adjusted to the individual weight) of VHC(DMSO:PEG200 1:1), positive control valproate (300 mg/kg), or TMC-120B(20, 10, 5, and 2.5 mg/kg) 30 min before electrical stimulation. VHCinjected mice showed characteristic seizure behavior with a meanduration of 36 seconds and a minimum duration of 17 seconds. Hence, micethat had seizures for less than 17 seconds were considered to beprotected. In line with previous studies, valproate treated mice werefully protected against the electrically-induced focal seizures as noneof the mice showed any seizure after electrical stimulation. Mice i.p.injected with 10 mg/kg TMC-120B had a significantly shorter seizureduration than the VHC control group (p≤01) with a mean of 10 seconds. Adose-dependent reduction in antiseizure activity was seen for miceinjected with 5 and 2.5 mg/kg TMC-120B (mean seizure duration of 23.5and 27 seconds, respectively), as well as with the higher dose of 20mg/kg (mean seizure duration of 27.5 seconds). The latter effect can beinfluenced by the poor solubility of 20 mg/kg TMC-120A in the solvent incontrast to the lower doses. Thus, the antiseizure activity of TMC-120Bas observed in the larval zebrafish PTZ seizure model translates to astandard mouse model of drug-resistant focal seizures, therebydemonstrating the effectiveness of our zebrafish-based ASD discoveryapproach and the potential of marine NPs. Moreover, these observationsconfirm the translation from zebrafish larvae to mice in the field ofepilepsy, as previously published [Patel and Patel (2011) J. App. Pharm.Sci. 1, 167-171; Perez et al. (2015) J Biomol Screen 20, 254-264].

Example 8 TMC-120A and TMC-120B Ameliorate Focal Seizures in the Mouse6-Hz (44 mA) Psychomotor Seizure Model

Despite the high genetic, physiological and pharmacologicalconservation, zebrafish are more distinct from humans than mammals[MacRae & Peterson (2015) Nat Rev Drug Discov, 14, 721-731; Wilcox etal. (2013) Epilepsia 54 S4, 24-34]. Therefore, we wanted to investigatewhether the antiseizure action of TMC-120A and TMC-120B observed in thelarval zebrafish model translates to a standard rodent seizure model.From the available rodent seizure models we chose the mouse 6-Hz (44 mA)psychomotor seizure model, a gold standard in current ASD discoveryefforts that is useful for screening and can detect compounds with novelantiseizure mechanisms and with potential against drug-resistantseizures [Wilcox et al (2013) cited above; Barton (2001) Epilepsy Res47(3), 217-227; Kehne et al. Neurochem Res (2017)]. It is an acute modelof drug-resistant focal impaired awareness seizures [Fisher et al.(2017) Epilepsia 58 (4), 531-542], previously referred to as complexpartial or psychomotor seizures [Holcomb & Dean in Psychomotor Seizures.In Encyclopedia of Child Behavior and Development, Goldstein, S.;Naglieri, J. A., Eds. Springer US: Boston, Mass., 2011; pp 1191-1192],that are induced by a low frequency, long duration corneal electricalstimulation [Kehne et al. (2017) Neurochem Res].

Male NMRI mice were intraperitoneally (i.p.) injected with a 50 μLvolume (adjusted to the individual weight) of VHC (DMSO:PEG200 1:1),positive control valproate (300 mg/kg), TMC-120A (10, 5, 2.5, and 1.25mg/kg), or TMC-120B (20, 10, 5, and 2.5 mg/kg) 30 min before electricalstimulation (FIG. 8). VHC injected mice showed characteristic seizurebehavior with a mean (±SD) duration of 32 seconds (s) (±13 s). In linewith previous studies, mice that were injected with valproate were fullyprotected against the induced seizures [Barton, M. (2001) cited above;Orellana-Paucar, et al. PLoS One 2013, 8 (12), e81634] as none of themice showed any seizure after electrical stimulation (p<0.001). Micei.p. injected with TMC-120A had a shorter seizure duration than the VHCcontrol group, which was significant at 10 mg/kg (p<0.05, mean durationof 17 s (±10 s)), 2.5 mg/kg (p<0.01, mean duration of 13 s (±6 s)), and1.25 mg/kg (p<0.05, mean duration of 15 s (±4 s)), but not at 5 mg/kg(mean duration of 20 s (±15 s)). Mice i.p. injected with TMC-120B alsohad a shorter seizure duration than the VHC control group, which wassignificant at 10 mg/kg (p<0.001, mean duration of 10 s (±8 s)). Anon-significant dose-dependent reduction in seizure duration was seenfor mice injected with 5 and 2.5 mg/kg TMC-120B (mean duration of 23.5 s(±11 s) and 27 s (±11 s), respectively). Finally, at the higher dose of20 mg/kg also a non-significant reduction in seizure duration was seenwith a mean seizure duration of 27.5 s (±20 s). The latter effect can beinfluenced by the poor solubility of 20 mg/kg TMC-120B in the solvent incontrast to the lower doses. Thus, the antiseizure activity of TMC-120Aand TMC-120B that was observed in the larval zebrafish PTZ seizure modeltranslates to a standard mouse model of drug-resistant focal seizures,thereby demonstrating the effectiveness of our zebrafish-based ASDdiscovery approach and the potential of marine NPs. Moreover, theseobservations confirm the translation of findings from zebrafish larvaeto mice in the field of epilepsy, as previously published [Buenafe, etal. 2013) ACS Chem Neurosci 4(11), 1479-1487; Orellana-Paucar et al.(2012) Epilepsy Behav 24 (1), 14-22.

1. An isoquinoline or 1H-2-Benzopyran, selected from the groupconsisting of TMC-120A, TMC-120B, TMC-120C, penicisochroman G,ustusorane B, compound 6 (7-methylfuro[3,2-h]isoquinoline-3(2H)-one) andcompound 7(2-(7-methyl-2,3-dihydrofuro[3,2-h]isoquinoline-2-yl)-propan-2-ol) foruse in the treatment or prevention of epilepsy.
 2. An isoquinoline or1H-2-Benzopyran in accordance with claim 1, in combination with halimideor plinabulin, for use in the treatment or prevention of epilepsy.
 3. Amethod for identifying pharmaceutical compounds against epilepsy, themethod comprising the steps of: providing a compound which comprises abenzene ring fused to a) a pyridine ring b) or a puran ring and fused toa modified or unmodified furan group as depicted in formula 1 or formula2 and,

testing the compound for antiseizure activity.
 4. The method accordingto claim 3, wherein said pyridine or puran ring is further methylated asdepicted in formula 3 or formula 4


5. The method according to claim 3, wherein said compound comprises amoiety as depicted in formula 5 or 6


6. The method according to claim 3, which comprises an isoquinoline or1H-2-Benzopyran moiety and testing the compound for antiseizureactivity.
 7. The method according to claim 3, wherein the compound witha isoquinoline or 1H-2-Benzopyran moiety is a TMC-120A, TMC-120B,TMC-120C, penicisochroman G, ustusorane B,furo[3,2-h]isoquinoline-3(2H)-one,7-methyl- andfuro[3,2-h]isoquinoline-2-methanol,2,3-dihydro-α,α,7-trimethyl-,(2S)—.8. The method according to claim 3, wherein halimide is added to saidcompound for testing antiseizure activity.
 9. The method according toclaim 3, wherein the isoquinoline or 1H-2-Benzypuran is a compound asdepicted in FIGS. 1 and/or 2, with modified molecular structure orstereochemistry.
 10. The method according to claim 3, whereinanti-seizure activity is determined in a zebrafish model.
 11. The methodaccording to claim 3, wherein anti-seizure activity is furtherdetermined in a mammalian model.
 12. The method according to claim 3,further comprising the step of testing the compound for a side effect.13. The method according to claim 3, further comprising the step offormulating a compound with determined anti-seizure activity into apharmaceutical composition with an acceptable carrier, for use in thetreatment of epilepsy.